Loss of Mitochondrial Tumor Suppressor Genes Expression Is Associated with Unfavorable Clinical Outcome in Head and Neck Squamous Cell Carcinoma: Data from Retrospective Study

<div><p>Mitochondrial genes play important roles in cellular energy metabolism, free radical generation, and apoptosis. Dysregulation of these genes have long been suspected to contribute to the generation of reactive oxygen species (ROS), increased proliferation and progression of cancer. A family of orthologues of yeast silent information regulator 3 (SIRT3), 4 (SIRT4) and mitochondrial tumor suppressor 1 (MTUS1) are important mitochondrial tumor suppressor genes which play an important role in the progression of multiple cancers. However, their role in the development of oxidative stress, enhanced proliferation and progression of head and neck squamous cell carcinoma (HNSCC) has not yet been studied. In this study we aimed to test the association between reduced mitochondrial tumor suppressor genes’ activities and enhancement in tissue oxidative stress and cell proliferation in HNSCC cases. The expression of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1), mitochondrial DNA repair gene (OGG1-2a) and a proliferation marker (Ki-67) was studied in a study cohort of 120 HNSCC patients and controls with reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR (qPCR) in order to determine the potential prognostic significance of these genes. A statistically significant downregulation of SIRT3 (p<0.001), SIRT4 (p<0.0001), MTUS1 (p<0.002) and OGG1 (p<0.0001) was observed in HNSCC compared to control samples. Ki-67 was also overexpressed (p<0.0001) in HNSCC versus control samples. Additionally, to explore gene–gene relationship, we observed a positive spearmen correlation between SIRT3 versus SIRT4 (r = 0.523***, p<0.0001), SIRT3 versus MTUS1 (r = 0.273***, p<0.001), SIRT3 versus OGG1-2a (r = 0.213*, p<0.03), SIRT4 versus OGG1 (r = 0.338***, p<0.0001) and MTUS1 versus OGG1-2a (r = 0.215*, p<0.03) in HNSCC cases. A negative spearman correlation was observed between OGG1 versus Ki-67 (r = -0.224**, p<0.01) and OGG1-2a versus Ki-67 (r = -0.224**, p<0.01) in HNSCC cases. Here we report that the deregulation of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1) in relation to decreased expression of mitochondrial DNA repair gene OGG1-2a and increased proliferation (measured by proliferation marker Ki-67) may be considered important factors in the development of head and neck squamous cell carcinoma.</p></div

Correlations between mitochondrial tumor suppressor genes (<i>SIRT3</i>, <i>SIRT4</i>, <i>MTUS1</i>), mitochondrial DNA repair gene (<i>OGG1-2a</i>), proliferation marker (<i>Ki-67</i>) expression and clinicopathological characteristics of primary HNSCC^†.

<p>Correlations between mitochondrial tumor suppressor genes (<i>SIRT3</i>, <i>SIRT4</i>, <i>MTUS1</i>), mitochondrial DNA repair gene (<i>OGG1-2a</i>), proliferation marker (<i>Ki-67</i>) expression and clinicopathological characteristics of primary HNSCC<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146948#t002fn001" target="_blank">^†</a>.</p

mRNA expression of SIRT4 in HNSCC cases of study cohort.

<p>Box plot comparing the SIRT4 mRNA levels of HNSCC and normal control samples, HNSCC samples with clinical stage I–II and clinical stage III–IV, with different T stages, with lymph node (N1–N2) and without lymph node (N0), with metastasis (M1-M2) and without metastasis (M0), with survival status and HNSCC samples with different grades. The p-values were computed using one-way analysis of variance and Tukey s’ post hoc test.</p

mRNA expression of MTUS1 in HNSCC cases of study cohort.

<p>Box plot comparing the MTUS1 mRNA levels of HNSCC and normal control samples, HNSCC samples with clinical stage I–II and clinical stage III–IV, with different T stages, with lymph node (N1–N2) and without lymph node (N0), with metastasis (M1-M2) and without metastasis (M0), with survival status and HNSCC samples with different grades. The p-values were computed using one-way analysis of variance and Tukey s’ post hoc test.</p

mRNA expression of Ki-67 in HNSCC cases of study cohort.

<p>Box plot comparing the Ki-67 mRNA levels of HNSCC and normal control samples, HNSCC samples with clinical stage I–II and clinical stage III–IV, with different T stages, with lymph node (N1–N2) and without lymph node (N0), with metastasis (M1-M2) and without metastasis (M0), with survival status and HNSCC samples with different grades. The p-values were computed using one-way analysis of variance and Tukey s’ post hoc test.</p

Association of expression deregulation of SIRT3, SIRT4, MTUS1, OGG1-2a and Ki-67 in HNSCC and lymph node and metastasis.

<p>Association of expression deregulation of SIRT3, SIRT4, MTUS1, OGG1-2a and Ki-67 in HNSCC and lymph node and metastasis.</p

Promoter Methylation Status Modulate the Expression of Tumor Suppressor (RbL2/p130) Gene in Breast Cancer

<div><p>Background</p><p>Aberrant expression of tumor suppressor genes may correspond to the abnormal cell development and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive.</p><p>Methods</p><p>In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expression was analyzed by quantitative real time PCR (syber green method). Promoter methylation status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests.</p><p>Results</p><p>We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as compared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methylation (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This highlights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis.</p></div

Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).

<p>Map of Rbl2/p130 promoter showing methylated and un-methylated CpG’s cytosine ranging from −10 to +80 with reference to TSS (transcription start site).</p

Rbl2/p130 promoter methylation and transcript expression analysis among various histological types of breast cancer.

<p><b>(A)</b> Relative expression of Rbl2/p130 in control and tumor tissues among various histological types of breast cancer. <b>(B)</b> Change in promoter methylation (ΔMeth) status in control and diseased tissues among different histological different types of breast cancer. <b>DCI;</b> ductal carcinoma in situ. <b>IDC;</b> invasive ductal carcinoma. <b>ILC;</b> invasive lobular carcinoma.</p

Mean comparison of Rbl2/p130 promoter methylation status using tukey test.

<p>Mean comparison of Rbl2/p130 promoter methylation status using tukey test.</p